Solid support reagents for the synthesis of 3&#39;-Nitrogen containing polynucleotides

ABSTRACT

The compounds are exemplified by the class of sulfoethyl oxygen-substituted carbamates, such compounds being useful as support reagents for automated polynucleotide synthesis of 3&#39;-nitrogen functionalized polynucleotides. The invention includes, in one aspect, a polynucleotide synthesis reagent having the structure: ##STR1## where T is an acid-cleavable hydroxyl protecting group, e.g., 4,4&#39;-dimethoxytritil; Q is a linker connecting the nitrogen and oxygen, e.g., n-hexyl; R 1  is a nitrogen substituent, e.g., hydrogen; R 2  through R 4  are separately hydrogen or lower alkyl; Y is an atom which is electronegative with respect to carbon, e.g., oxygen or sulfur; X 1  is an atom which is electronegative with respect to carbon, e.g., sulfone; Z is a bond or spacer arm, e.g., ethylsuccinate; and W is a derivatized solid synthesis support capable of linking to Z, e.g., an amino-dirivitized controlled pore glass. In a second aspect, the present invention includes a polynucleotide synthesis reagent having the structure: ##STR2## where T, Q, R 1 , R 2  -R 3 , X 2 , Z, and W are as defined above.

BACKGROUND

This invention relates generally to solid support reagents used for thesynthesis of functionalized polynucleotides, and more particularly, tothe synthesis of polynucleotides having a nitrogen atom located at the3'-end.

The continued rapid development of non-isotopic polynucleotide probes,DNA/RNA amplification methods, and bioactive antisense and triplexsynthetic reagents, has greatly increased the demand for chemicallymodified polynucleotides. One popular approach to polynucleotidemodification is to introduce a primary aliphatic amine to one end of thepolynucleotide, thereby making it possible to readily functionalize thepolynucleotide with substituents containing electrophilic moieties,e.g., isothiocyanates or activated esters. Common substituents includefluorophores, enzymes, biotin, intercalators, cross-linkers, nucleicacid cleaving reagents, modifiers of cellular uptake, and the like.

The most effective and convenient method for the introduction of annitrogen atom to an end of a synthetic polynucleotide is to use anappropriately fuctionalized synthesis support followed by selectivecleavage of the nitrogen-functionalized polynucleotide from thatsupport. A number of methods currently exist for synthesizing3'-nitrogen functionalized polynucleotides using modified supports,however, all of these methods produce a racemic mixture of productsand/or require non-standard automated polynucleotide synthesis reagentsand/or procedures, thereby complicating the purification and/orsynthesis of these compounds.

For the foregoing reasons, there is a need for a solid support reagentcapable of synthesizing 3'-nitrogen functionalized polynucleotides innon-racemic preparations using standard polynucleotide synthesisreagents, systems and procedures. More specifically, a modified solidsupport which is stable to (i) polynucleotide synthesis capping reagentssuch as acetic anhydride and pyridine, (ii) oxidants such as iodine,(iii) medium-strength acids such as trichloroacetic acid, and (iv)phosphorylating agents such as phosphoramidites; while at the same timebeing labile to typical polynucleotide synthesis cleavage reagents suchas ammonium hydroxide.

SUMMARY

The present invention is directed toward our discovery of apolynucleotide synthesis support for use in automated polynucleotidesynthesis that is useful for synthesizing 3'-nitrogen functionalizedpolynucleotides.

It is an object of the invention to provide a solid support reagent thatis capable of supporting the synthesis of polynucleotides in non-racemicpreparations using standard polynucleotide synthesis reagents, systemsand procedures.

The present invention includes, in one aspect, a polynucleotidesynthesis reagent comprising a compound of the formula: ##STR3## wherethe variable elements of the above formula are defined as follows: T isan acid-cleavable hydroxyl protecting group; Q is a linker connectingthe nitrogen and oxygen; R₁ is an inert nitrogen substituent; R₂ throughR₄ are separately hydrogen or lower alkyl; Y is an atom which iselectronegative with respect to carbon; X₁ is an atom which iselectronegative with respect to carbon; Z is a bond or spacer arm; and Wis a derivatized solid synthesis support capable of joining to Z.

In a second aspect, the present invention includes a polynucleotidesynthesis reagent comprising a compound of the formula: ##STR4## wherethe variable elements of the above formula are defined as follows: T isan acid-cleavable hydroxyl protecting group; Q is a linker connectingthe nitrogen and oxygen; R₁ is an inert nitrogen substituent; R₂ throughR₄ are separately hydrogen or lower alkyl; Y is an atom which iselectronegative with respect to carbon; X₂ is an atom which iselectronegative with respect to carbon; Z is a bond or spacer arm; W isa solid synthesis support capable of joining to Z.

In one preferred embodiment of either aspect, T is 4,4'-dimethoxytrityl,monomethoxytrityl, α-naphthyldiphenylmethyl, ortri(p-methoxyphenyl)methyl. More preferably, T is 4,4'-dimethoxytrityl.

In another preferred embodiment of either aspect, Q is lower alkyl,lower alkylene oxide, or, amide, carbamate, sulfonamide, or urea when incombination with a nitrogen of the solid support reagent, or anycombination thereof. More preferably, Q is lower alkyl or lower alkyleneoxide.

In yet another preferred embodiment of either aspect, R₂ through R₄ arehydrogen.

In another preferred embodiment of either aspect, Y is oxygen or sulfur.More preferably, Y is oxygen.

In a preferred embodiment of the first aspect of the invention, X₁ issulphonyl, carbonyl, sulfoxide, perfluoro lower alkyl, or sulfonyl-,carbonyl-, sulfoxide-, nitro-, cyano-, or perfluoro loweralkyl-substituted aryl. More preferably, X₁ is sulphonyl, carbonyl,sulfoxide.

In a preferred embodiment of the second aspect of the invention, X₂ issulphonyl, carbonyl, sulfoxide, cyano, perfluoro lower alkyl, orsulfonyl-, carbonyl-, sulfoxide-, nitro-, cyano-, or perfluoro loweralkyl-substituted aryl. More preferably, X₂ is sulphonyl, carbonyl, orcyano.

In another preferred embodiment of either aspect, Z is, in combinationwith a terminal nitrogen of the solid synthesis support derivatized witha nitrogen-terminated linker, carbamate, urea, amide, sulfonamide, or agroup of the formula: ##STR5## wherein v is between 0 and 20.

In another preferred embodiment of either aspect, W is CPG derivatizedwith an amino-terminated linker.

In a final preferred embodiment of either aspect, W is porouspolystyrene derivatized with an amino-terminated linker.

In a preferred embodiment of the first aspect, the polynucleotidesynthesis reagent is a compound of the formula ##STR6##

In a preferred embodiment of the second aspect, the polynucleotidesynthesis reagent is a compound of the formula ##STR7## where in both ofthe above structures, T is 4,4'-dimethoxytrityl (DMT), Q is n-hexyl, R₁-R₄ is hydrogen, Y is oxygen, X₁ (or X₂) is sulfonyl, Z is an ethylsuccinate linker, and W is an amino derivatized solid support.

In a third aspect, the present invention includes a method for thesynthesis of polynucleotides containing a 3'-nitrogen atom using thesolid supports of the present invention. The synthesis takes place on asolid support reagent having the formula: ##STR8## where, T is anacid-cleavable hydroxyl protecting group; Q is a linker for connectingnitrogen and oxygen; R₁ is a nitrogen substituent; R₂ through R₄ areseparately hydrogen or lower alkyl; Y is an atom which iselectronegative with respect to carbon; X₁ is an atom which iselectronegative with respect to carbon; X₂ is an atom which iselectronegative with respect to carbon; Z is a bond or spacer arm; and Wis a derivatized solid synthesis support capable of linking to Z. Next,the solid support is treated with acid to remove the acid-cleavablehydroxyl protecting group. A protected nucleoside monomer is then addedalong with a weak acid, forming a linkage between the nucleoside and thegrowing support-bound chain. The unreacted sites on the solid supportare then capped with a capping reagent and oxidizing reagents are added.The above steps are the repeated until the polynucleotide chainelongation is complete. At this point, the oligonucleotide is stillbound to the solid support with protecting groups on the phosphates andthe exocyclic amines of the bases. The oligonucleotide is cleaved fromthe support by treatment with concentrated ammonium hydroxide, and theprotecting groups are removed by treating the crude DNA solution inammonium hydroxide at an elevated temperature, e.g., 55° C.

DESCRIPTION

I. DEFINITIONS:

The term "lower alkyl" as used herein denotes straight-chain,branched-chain, and cyclized alkyl groups containing from 1 to 8 carbonatoms.

The term "lower alkylene oxide" as used herein denotes straight-chain,branched-chain, and cyclized alkylene oxide groups containing from 2 to8 carbon atoms, e.g., polyethylene oxide.

The term "electron withdrawing" denotes the tendency of a substituent toattract valence electrons from neighboring atoms, i.e., the substituentis electronegative with respect to neighboring atoms. One popular andwell accepted index of electronegativity is the Pauling index.

II. DETAILED DESCRIPTION:

Reference will now be made in detail to the preferred embodiments of theinvention. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to these embodiments. On the contrary, theinvention is intended to cover alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

In a first preferred embodiment, the solid support of the presentinvention is defined by the formula: ##STR9## where T refers generallyto an acid-cleavable hydroxyl protecting group. Preferably, T is thetriphenylmethyl radical and its electron-donating-substitutedderivatives, where, as used herein, the term "electron-donating" denotesthe tendency of a substituent to release valence electrons toneighboring atoms in the molecule of which it is a part, i.e., it iselectropositive with respect to neighboring atoms in the molecule.Preferably, electron-donating substituents include amino, lower alkylhaving between 1 and 8 carbon atoms, lower aryl having between 1 and 8carbon atoms, alkoxy having from 1 to 8 carbon atoms, and the like. Morepreferably, the electron-donating substituents are methoxy. Exemplarytrityls include 4,4'-dimethoxytrityl (i.e. bis(p-anisyl)phenylmethyl),monomethoxytrityl, α-naphthyldiphenylmethyl, tri(p-methoxyphenyl)methyl,and the like. Attachment and cleavage conditions for these and othertrityls can be found in Greene and Wuts, Protective Groups in OrganicSynthesis, 2nd Edition (John Wiley, New York, 1991).

Q is a linker which, when the 3'-nitrogen-functionalized oligonucleotideis cleaved from the support, serves to link the 3'-nitrogen with theoligonucleotide through an oxygen. In some cases, Q also serves toprovide a degree of spatial separation between the oligonucleotide andthe 3'-nitrogen, e.g., to increase the reactivity of the 3'-nitrogen byreducing the steric hindrance caused by the oligonucleotide. Finally, Qmay add functionality to the 3'-nitrogen oligonucleotide such asenhanced nuclease resistance, solubility, transport properties,hybridization, altered electrophoretic mobility, and the like. Q shouldbe stable to typical DNA synthesis reagents. Because Q is not a centralfeature of the invention and provides a generic function, it will beappreciated that Q can have a wide variety of forms. Preferably, Q islower alkyl, lower alkylene oxide, or, amide, carbamate, sulfonamide, orurea when in combination with a nitrogen of the solid support reagent,or any combination thereof. More preferably, Q is lower alkyl or loweralkylene oxide.

R₁ is a nitrogen substituent which can vary greatly depending on thenature of the desired final product. It will be appreciated that becauseR₁ is not a central feature of the invention and provides a genericfunction, R₁ can have a wide variety of forms. R₁ is chosen so that thebonded nitrogen atom is chemically stable during synthesis andsubsequent to oligonucleotide cleavage. Preferably, R₁ is stable tostandard polynucleotide synthesis reagents and does not interfere withthe elimination of the Y═C═O group during polynucleotide cleavage. If areactive amino group is desired subsequent to polynucleotide cleavage,R₁ should not substantially interfere with the nitrogen reactivity. Inthis case, R₁ is preferably lower alkyl or hydrogen. Most preferably, R₁is hydrogen.

If a reactive amino group is not required in the final product, R₁ needonly be stable to standard polynucleotide synthesis chemistry and notinterfere with the elimination of the Y═C═O group during polynucleotidecleavage. Preferably, R₁ is lower alkylene oxide, hydrogen, alkyl,sulfonyl, acyl, alkoxycarbonyl, or carbamoyl. Alternatively, R₁ is afunctional moiety such as a dye, specific binding reagent, atransport-enhancing reagent, e.g., cholesterol, and the like.

Y is a functionality which serves to polarize the double bond betweenitself and the bonded carbon such that the bonded carbon is madeelectropositive, thereby favoring the elimination of a Y═C═O group uponbase-cleavage from the support. Preferably Y is electronegative withrespect to carbon. Preferably, Y is either oxygen or sulfur. Morepreferably, Y is oxygen.

X₁ is an electron withdrawing functionality which serves to make ahydrogen which is bonded to the same carbon as X₁ acidic, i.e., pKabetween 15 and 35, thereby facilitating elimination by ammonia.Preferably, X₁ is sulphonyl, carbonyl, sulfoxide, perfluoro lower alkyl,or sulfonyl-, carbonyl-, sulfoxide-, nitro-, cyano-, or perfluoro loweralkyl-substituted aryl. More preferably, X₁ is sulphonyl, carbonyl, orsulfoxide.

Z is a bond or spacer arm which serves to link the solid support and thefunctional regions of the invention. In many instances, Z also serves toprovide spatial separation between the solid support and the functionalregions of the invention in order to eliminate the transport resistancesassociated with solid phase synthesis, i.e., to allow theoligonucleotide synthesis to proceed with liquid-phase kinetics. Zshould be stable to typical DNA synthesis reagents. Preferably, Z is, incombination with the terminal nitrogen of the derivatized solidsynthesis support, carbamate, urea, amide, sulfonamide, or a group ofthe formula: ##STR10## where v is an integer between 0 and 20.

R₂ through R₄ are chosen so as to form a stable spacer betweenchemically active portions of the support. Preferably, R₂ through R₄each taken separately represent hydrogen or lower alkyl. Morepreferably, R₂ through R₄ taken separately each represent hydrogens.

W is a derivatized solid substrate on which the polynucleotide synthesistakes place. W can have a variety of forms and compositions, however,the solid substrate should: (i) be substantially insoluble in thereaction solvents (ii) be chemically stable to standard polynucleotidesynthesis reagents, (iii) be capable of chemical derivitization, (iv)provide the desired oligonucleotide loading, (v) have adequatecompression strength to withstand elevated pressure encountered duringprocessing, and, (vi) be available in a desirable particle size rangeand distribution. Furthermore, W is derivatized in order to facilitateattachment of the oligonucleotide to the support.

In one preferred embodiment, W is an inorganic polymer support. A widevariety of inorganic polymers can be employed in the present inventionand these include, for example, silica, porous glass, aluminosilicates,borosilicates, metal oxides such as alumina and nickel oxide, variousclays, and the like. Preferably, the inorganic solid substrate iscontrolled pore glass (CPG). Controlled pore glass consists of uniformlymilled and screened particles of almost pure silica that are honeycombedwith pores of a controlled size. It is manufactured from a borosilicatematerial that has been specially heat treated to separated the boratesfrom the silicates. The pores are formed by removing the borates by anacidic etching process, their size being dependent on the nature of theheating process. More preferably, the CPG is in the form of 150 μmdiameter particles having 500 Å pores, e.g., Users Manual Model 392 and394 polynucleotide Synthesizers, pages 6-5 through 6-9, AppliedBiosystems, Ver. 2.00, Doc. Rev. A, Part No. 902351 (1992).

Derivatization of CPG supports with amino-terminated linkers is wellknown in the art of polynucleotide synthesis, e.g., Gait, Editor,Oligonucleotide Synthesis, pages 45-49 (IRL Press, 1984), and in fact,CPG beads derivatized with an alkyl amine having a primary amino loadingof about 100 mmol/g are commercially available (Pierce Chemical Company,Rockford, Ill.). Briefly, in the case of alkyl amino substrates, asuspension of CPG particles is reacted with anaminoalkyltrimethoxysilane reagent, filtered, and dried.

A second preferred solid substrate is non-swellable porous polystyrene.As used herein, "non-swellable" means that the porous polystyrenematerial remains substantially mechanically rigid, in particular, doesnot appreciably increase in volume, when exposed to solvents, reactantsand products of the phosphoramidite and/or hydrogen phosphonatepolynucleotide synthesis chemistries. As used herein, "porous" meansthat the non-swellable polystyrene contains pores having substantiallyuniform diameters in the range of between 100 and 4000 Å.

The polystyrene support is amino-derivatized by standard procedures,e.g., Wallace et al., pages 638-639 in Scouten ed., Solid PhaseBiochemistry (John Wiley & Sons, 1980); Wright et al. Tet. Lett., 34:3373-3376 (1993); Bayer et al, U.S. Pat. No. 4,908,405; AppliedBiosystems Research News, Model 390Z, February 1994. Briefly,hydroxymethylpthalimide is reacted with the polystyrene support with acatalytic amount of methylsulfonic acid to form pthalimidomethylpolystyrene. This material is then treated with hydrazine to remove thepthalimide protecting group to give aminomethylated polystyrene.Typically, the amino loading varies from 20 to 60 μmoles of aminofunctionality per gram of non-swellable porous polystyrene. The loadinglevel can be controlled by adjusting the concentrations of the reagentsand reaction times.

A recently developed alternative polystyrene derivatizing chemistryreplaces the terminal amino group with a free hydroxyl group byattaching several polyoxyethylene residues or chains having freehydroxyl groups available for coupling with the polynucleotide, e.g.,Bayer and Rapp, U.S. Pat. No. 4,908,405; Gao et al., Tetrahedron Lett.,32(40):5477-5480 (1991).

In a third preferred embodiment, W is a non-polystyrene organic polymer.The polymer support can be derived from naturally occurring materialswhich are synthetically modified, and synthetic materials. Of particularinterest are polysaccharides, particularly crosslinked polysaccharides,such as agarose, which is available as Sepharose™, dextran, which isavailable as Sephadex™, cellulose, starch, and the like (Sepharose™ andSephadex™ being trademarked products of Pharmacia Fine Chemicals,Piscataway, N.J.). Other materials include polyacrylamides, polyvinylalcohols, silicones, Teflons™, and the like.

In a second preferred embodiment, the solid support of the presentinvention is defined by the formula: ##STR11## wherein X₂ is an electronwithdrawing functionality which serves to make any hydrogen which isbonded to the same carbon as X₂ acidic, thereby facilitating eliminationby ammonia. Preferably, X₂ is sulphonyl, carbonyl, sulfoxide, cyano,perfluoro lower alkyl, or sulfonyl-, carbonyl-, sulfoxide-, nitro-,cyano-, or perfluoro lower alkyl-substituted aryl. More preferably, X₂is sulfonyl, carbonyl, or cyano.

All other variable elements in the compounds of Formula II are asdefined as above in the context of Formula I compounds.

III. GENERAL SYNTHETIC METHOD:

A. Synthesis of the X1-containing solid support of Formula I:

The following is a preferred generalized synthesis method for thecompounds of Formula I. Generally, the reaction scheme involvespreparing a hydroxyl-protected alcoholamine (T-amine), preparing ahydroxyl-protected carbamatealcohol (T-CA) by reacting a diol with aphosgene equivalent and then the T-amine. The T-CA is then treated toform an active T-CA linker which is reacted with an amino-derivatizedsolid substrate.

To form the T-amine, first, the amine moiety of an aminoalcohol isprotected with a base-labile protecting group. The aminoalcohol (approx.1.0 equivalent), defined by the formula

    HOQNHR.sub.1

wherein the variable elements are as indicated above, is dissolved in anorganic solvent, e.g., methanol, ether, methylene chloride, and thelike, and a base-labile amino protecting reagent (approx. 1.1equivalents), e.g., ethyltrifluoroacetate, is added dropwise to theaminoalcohol solution at a temperature of between -5° and 25° C., andstirred for between 1-6 hrs at a temperature of between 0° and 40° C.,after which the solvent is evaporated under vacuum. Exemplary aminoalcohols which are commercially available include aminoethanol,6-amino-1-hexanol, aminocyclohexanol, 2-(2-aminoethoxy)ethanol,leucinol, and the like. The residue is dissolved in a water-immiscibleorganic solvent, e.g., methylene chloride, ether, ethylacetate, and thelike, washed with water, and dried over sodium sulfate. The solvent isthen evaporated under vacuum to give a protected aminoalcohol product.

Next, the alcohol moiety of the protected aminoalcohol is protected. Theprotected aminoalcohol (approx. 1.0 equivalent) and a tertiary amine(approx. 1.5 equivalents), e.g., diisopropylethylamine, triethylamine,and the like, are dissolved in an aprotic organic solvent, e.g.,methylene chloride, ether, and the like, and an acid-labile hydroxylprotecting agent, (approx. 1.1 equivalents), e.g., a tritylating agentsuch as dimethoxytritylchloride, is added at a temperature of between-10° and +10° C. The mixture is stirred at between 0° and 25° C. forbetween 5 and 25 hrs, after which it is diluted with an equal volume ofthe organic solvent used in the reaction. The reaction solution is thenwashed with a saturated sodium bicarbonate solution, dried over sodiumsulfate, and concentrated under vacuum to give a T-protectedaminoalcohol.

Finally, the amine moiety of the T-protected aminoalcohol is deprotectedby treatment with base. The T-protected aminoalcohol is dissolved in apolar organic solvent, e.g., methanol, a basic aqueous solution, e.g.,4N sodium hydroxide, is added at between 0° and 25° C., and the reactionis stirred at between 25° and 60° C. for between 10 min and 2 hrs. Theorganic solvent is evaporated under vacuum and the residue is dissolvedin a water-immiscible organic solvent, e.g., ethylacetate, ether, andthe like, the solution is washed with water, and dried over sodiumsulfate. The solvent is evaporated under vacuum to give a T-aminedefined by the formula:

    TOQNHR.sub.1

wherein the variable elements are as indicated above.

To form the T-CA, a dry diol is used having the formula:

    HOCR.sub.2 R.sub.3 CHR.sub.4 X.sub.1 Z'OH

wherein Z' is in some cases equivalent to Z, and in other cases is aprecursor to Z, depending on the method used to subsequently activatethe T-CA. The other variable elements are as indicated above. The drydiol (2 to 10 equivalents) and a tertiary amine (1.0 equivalent), e.g.,diisopropylethylamine, are dissolved in an aprotic organic solvent,e.g., pyridine, a phosgene equivalent (1.0 equivalent), e.g.,4-nitrophenylchloroformate, is added between 0° and 25° C., and thesolution is stirred at room temperature for between 10 min and 2 hrs.This solution is then added to a solution of the T-amine prepared above(approx. 0.25 to 1 equivalent) along with a tertiary amine (approx. 1.0equivalent), e.g., diisopropylethylamine, triethylamine, and the like,in an aprotic solvent, e.g., pyridine. The reaction is stirred atbetween 0° and 25° C. for between 10 min and 2 hrs, the solvent isevaporated under vacuum, the residue is dissolved in a water-immiscibleorganic solvent, e.g., ethylacetate, washed with water, and dried oversodium sulfate. The solvent is evaporated under vacuum to give aT-carbamate alcohol (T-CA) product defined by the formula: ##STR12##wherein the variable elements are as indicated above.

The above T-CA is then converted to an active linker using either one oftwo preferred procedures. In the first preferred procedure, the T-CA istreated with an amine base, e.g., 4-dimethylaminopyridine, and ananhydride, e.g., succinic anhydride, in an aprotic solvent, e.g.,methylene chloride, for between 10 and 60 min at between 10° and 60° C.The solution is washed with a weak aqueous acid, e.g., citric acid,dried over sodium sulfate, and concentrated under vacuum to give aT-carbamate alcohol linker (T-CA linker). The T-CA linker is thenactivated by treatment with an equimolar solution of1-hydroxybenzotriazole (HOBT) and2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium (HBTU) (0.45Msolution in N,N-dimethylformamide) in a polar aprotic solvent, e.g.,dimethyl formamide, followed by the addition of a tertiary amine, e.g.,diisopropylethyl amine. The reaction is stirred at between 5° and 35° C.for between 10 and 60 min to give an active T-CA linker.

In the second preferred activating procedure, the T-CA is treated with aphosgene equivalent (approx. 1.1 equivalent), e.g.,4-nitrophenylchloroformate, and a tertiary amine (approx. 1.1equivalent), e.g., diisopropylethylamine, in an aprotic solvent, e.g.,pyridine, at between 0° and 25° C. for between 10 min and 2 hrs to givethe active T-CA linker.

Finally, to attach the active T-CA linker to a solid substrate having anamino-terminated linker, e.g., an aminoalkyl solid substrate, e.g.,aminopropyl CPG, aminopropyl polystyrene, aminoalkylpolyethyleneglycolpolystyrene, and the like, is added to the active T-CA linker andallowed to react at between 5° and 40° C. for between 1 and 10 hrs withoccasional shaking or swirling of the reaction vessel. The derivatizedsolid support product is then filtered and washed in an organic solvent,e.g., methylene chloride, treated with a capping reagent (1:1:1 v/v/vacetic anhydride:N-methylimidazole:lutidine) for between 10 min and 2hrs, filtered, washed with an organic solvent, and dried under vacuum togive the solid support reagent of Formula I as a white solid.

B. Alternative convergent synthesis of the X₁ -containing solid supportof Formula I:

The following is a preferred alternative generalized synthesis methodfor the compounds of Formula I. A diol as above (1 to 5 equivalents) anda tertiary amine (1.1 equivalents), e.g., triethylamine, are dissolvedin an aprotic organic solvent, e.g., methylene chloride, and anacid-labile alcohol protecting reagent (1 equivalent), e.g.,dimethoxytritylchloride, is added and stirred at between 0° and 25° C.for between 10 min and 5 hrs. The solution is washed with water anddried over sodium sulfate to give a monoprotected diol.

The monoprotected diol is then transformed to an active linker andattached to an aminoalkyl solid substrate similar to the T-CA in MethodA above to give a T-linker support.

The T-linker support is then deprotected with acid, e.g.,trichloroacetic acid in methylene chloride, at between 10° and 30° C.for between 1 and 30 min, washed with an organic solvent, e.g.,methylene chloride, and dried, resulting in an alcohol-linker support.

The alcohol linker support is treated with a phosgene equivalent, e.g.,4-nitrophenylchloroformate, and a tertiary amine, e.g.,diisopropylethylamine, in an aprotic organic solvent, e.g., pyridine,methylene chloride, acetonitrile, and the like, at between 10° and 30°C. for between 5 and 60 min. The support is then washed with an aproticorganic solvent, e.g., methylene chloride, to give a carbonate linkersupport.

The carbonate linker support is then treated with a T-amine (see MethodA above) in a basic organic solvent, e.g., pyridine in acetonitrile,triethylamine in methylene chloride, and the like, at between 10° and30° C. for between 10 min and 2 hrs. The support is washed with anaprotic organic solvent, e.g., methylene chloride, treated with apolynucleotide synthesis capping reagent (see Method A above) forbetween 10 min and 2 hrs, washed with an aprotic organic solvent, anddried to give the solid support of Formula I.

C. Synthesis of the X₂ -containing solid support of Formula II:

The following is a preferred generalized synthesis method for thecompounds of Formula II. The starting material is a X₂ linker defined bythe formula: ##STR13## wherein Z" is a precursor to Z and the othervariable elements are as indicated above.

If the X₂ linker contains an amine, alcohol, or thiol, it is firstprotected with a non-base labile and non-acid labile protecting group,e.g., benzyl or silyl. Protocols for use of these and other applicableprotecting groups can be found elsewhere, e.g., Greene and Wuts,Protective Groups in Organic Synthesis, 2nd Edition (John Wiley, NewYork, 1991).

The X₂ linker is then converted to an alcohol by treating with a strongbase (2 equivalents), e.g., lithium diisopropylamide, sodium hydride,and the like, in a dry polar aprotic solvent, e.g., dimethylformamide,with stirring under argon. A ketone or aldehyde (1 equivalent) in a drypolar aprotic solvent is then added dropwise at between -40° and 0° C.,the reaction is stirred at between -40° and 25° C. for between 10 minand 2 hrs, the reaction is quenched with water and concentrated byevaporation under vacuum. The residue is then dissolved in a waterimmiscible solvent, e.g., ethyl acetate, ether, methylene chloride, andthe like, washed with water, dried over sodium sulfate, and concentratedunder vacuum to give an alcohol linker defined by the formula: ##STR14##wherein the variable elements are as indicated above.

The alcohol linker is then activated according to the above secondpreferred activating procedure for the active T-CA linker in Method Aabove to give the activated alcohol linker.

The T-amine (1 equivalent) and a tertiary amine (1 equivalent), e.g.,diisopropylethylamine, is added and stirred at between 0° and 25° C. forbetween 10 min and 2 hrs. The solvent is evaporated under vacuum and theresidue is dissolved in a water immiscible solvent, e.g., ethyl acetate.The solution is washed with water and dried over sodium sulfate. Thesolvent is then evaporated under vacuum to give the T-carbamate linker.

If the product contains a benzyl-protected amine, alcohol, or thiol, itis deprotected by hydrogenolysis, e.g., using hydrogen in combinationwith a palladium catalyst. Alternatively, if the product contains asilyl-protected amine, alcohol, or thiol, it is deprotected by treatmentwith a fluoride reagent, e.g., tetrabutylammonium fluoride.

If the T-carbamate linker is an amine, alcohol or thiol, then it can beactivated as described above for the active T-CA linker in Method A. Ifthe T-carbamate linker is a carboxylic or sulfonic acid, then it can beactivated by treatment with a HOBT/HBTU reagent as described above. Theresulting activated T-carbamate linker is then reacted with anaminoalkyl solid substrate as above to give the solid support shown inFormula II.

IV. UTILITY:

A preferred utility of the solid support of the present invention is inthe synthesis of polynucleotides containing a nitrogen atom located atits 3'-end. Detailed descriptions of the chemistry used to formpolynucleotides are provided elsewhere, e.g., Caruthers et al., U.S.Pat. No. 4,458,066; Caruthers et al., U.S. Pat. No. 4,415,732; Carutherset al., Genetic Engineering, 4: 1-17 (1982); Users Manual Model 392 and394 Polynucleotide Synthesizers, pages 6-1 through 6-22, AppliedBiosystems, Part No. 901237 (1991). Accordingly, these references areincorporated by reference for those descriptions.

The phosphoramidite method of polynucleotide synthesis is the preferredmethod because of efficient and rapid coupling and the stability of thestarting materials. The synthesis is performed with the growingpolynucleotide chain attached to a solid substrate, so that excessreagents, which are in the liquid phase, can be easily removed byfiltration, thereby eliminating the need for purification steps betweencycles.

The following briefly describes the steps of a typical polynucleotidesynthesis. The first step of the synthesis cycle is treatment of thesolid support with acid to remove the hydroxyl protecting group, freeingthe hydroxyl for the subsequent coupling reaction. A activatedintermediate is then formed by simultaneously adding the phosphoramiditenucleoside monomer and a weak acid, e.g., tetrazole, and the like, tothe reaction. The weak acid protonates the nitrogen of thephosphoramidite forming a reactive intermediate. This intermediate is soreactive that addition is complete within 30 s. The next step, capping,terminates any polynucleotide chains that did not undergo addition.Capping is preferably done with acetic anhydride and 1-methylimidazole.Finally, the internucleotide linkage is converted from the phosphite tothe more stable phosphotriester. Iodine is used as the preferredoxidizing agent and water as the oxygen donor. After oxidation, thehydroxyl protecting group is removed with a protic acid, e.g.,trichloroacetic acid or dichloroacetic acid, and the cycle is repeateduntil chain elongation is complete. After synthesis, the polynucleotidechain is cleaved from the support using a base, e.g., ammoniumhydroxide. Ammonia treatment also removes the cyanoethyl phosphateprotecting groups. Finally, the protecting groups on the exocyclicamines of the bases are removed by treating the polynucleotide solutionin ammonium hydroxide at an elevated temperature, e.g., 55° C.

It will be apparent to those skilled in the art of polynucleotidesynthesis that the present invention can also be used in conjunctionwith other synthetic methods, e.g., hydrogen phosponate orphosphotriester chemistries.

V. EXAMPLES

The following examples are intended to illustrate the preparation andapplication of the solid support reagents of the present invention. Thevalues of the parameters used are only intended to exemplify theinvention and are not to be considered limitations thereof.

Example 1 Synthesis of N-trifiuoroacetyl-6-amino-1-hexanol

6-amino-1-hexanol (179 g) (Aldrich Chemical Company, Inc., Milwaukee,Wis.) was dissolved in methanol (358 ml) and ethyltrifluoroacetate (239g) (Aldrich) was added dropwise to the solution over a period of 20 min.After stirring the reaction for 2.5 hrs, the solvent was removed undervacuum and the residue was dissolved in methylene chloride (250 ml),whereupon the solution was washed with water (3×300 ml) and a saturatedsodium chloride solution (200 ml), and dried over sodium sulfate.Finally, the solvent was removed under vacuum giving a white solid (299g).

Thin layer chromatography (TLC) Analysis: A TLC plate (Silica Gel GF,250 μm thickness, 10×20 cm scored plates, Analtech, Inc., Newark Del.)was developed with 100% ethylacetate and stained with 5% phosphomolybdicacid in isopropyl alcohol. The R_(f) of the 6-amino-1-hexanol and theN-trifluoroacetyl-6-amino-1-hexanol was 0 and 0.25, respectively.

Example 2 Synthesis of1-0-(4,4'-dimethoxytrityl)-N-trifluoroacetyl-6-aminohexane

The N-trifluoroacetyl-6-amino-1-hexanol (10 g) from Example 1 anddiisopropylethylamine (12.1 g) (Aldrich) were dissolved in methylenechloride (100 ml), ice-cooled to 5° C. and dimethoxytritylchloride (17.5g) (Aldrich) was added to the cooled mixture. The mixture was stirredovernight (15 hr) during which the temperature was maintained at 5° C.for the first hour then allowed to rise to room temperature thereafter.Methylene chloride (100 ml) was added and the mixture was washed with asaturated sodium bicarbonate solution (100 ml) followed by a saturatedsodium chloride solution (100 ml). The mixture was then dried oversodium sulfate and the solvent was removed under vacuum to give thedesired product (29 g).

TLC Analysis: A TLC plate (same type as above) was developed with 50%ethyl acetate 1% triethylamine in hexane. The R_(f) of the1-0-(4,4'-dimethoxytrityl)-N-trifluoroacetyl-6-aminohexane was 0.9.

Example 3 Synthesis of 1-0-(4,4'-dimethoxytrityl)-6-aminohexane

The 1-0-(4,4'-dimethoxytrityl)-N-trifluoroacetyl-6-aminohexane (20.9 g)from Example 2 was dissolved in methanol (100 ml), ice-cooled, and 4Nsodium hydroxide was added (16.6 ml). The reaction was allowed to warmto room temperature, heated to 50° C. for 10 min using a heat gun, thenstirred overnight (15 hrs) at room temperature. The methanol was removedunder vacuum and the residue was mixed with water (100 ml) and ethylacetate (150 ml). The organic layer was then washed with saturatedaqueous sodium chloride (2×100 ml) and dried over sodium sulfate. Thesolvent was removed under vacuum to give the product as an oil (16.9 g).

Example 4 Synthesis of the carbamate adduct of1-0-(4,4'-dimethoxytrityl)-6-aminohexane and 2,2'-sulfonyldiethanol

Prior to the synthesis of the carbamate adduct of1-0-(4,4'-dimethoxytrityl)-6-aminohexane and 2,2'-sulfonyldiethanol,2,2'-sulfonyldiethanol (Aldrich) was dried using the followingprocedure. 2,2'-sulfonyldiethanol (200 g of a 65% aqueous solution) wasmixed with acetonitrile (200 ml), the mixture was distilled, anddistillate was collected (240 ml) over a boiling point range of 70° C.to 90° C. The still pot was cooled to room temperature, additionalacetonitrile was added (200 ml), the distillation process was repeated,and additional distillate was collected (270 ml). The distillation headwas then replaced by a Stark trap, toluene (150 ml) was added, and themixture was brought to reflux. After 3 hrs of trapping, water wasrecovered (12 ml), and the solvent removed under vacuum to give aviscous oil (105 g).

The dried 2,2'-sulfonyldiethanol (14.7 g) was then dissolved intetrahydrofuran (100 ml) and stripped to dryness by rotary evaporation.The residue was dissolved in pyridine (100 ml) and diisopropylethylamine(12.3 g) under argon and cooled to approximately 10° C. using an icebath, after which 4-nitrophenylchloroformate (9.6 g) (Aldrich) was addedto the stirring solution and the reaction was allowed to warm toapproximately 25° C. After 45 min, the reaction was cooled to 15° C. andthe 1-0-(4,4'-dimethoxytrityl)-6-aminohexane (5 g) from Example 3 wasadded. After 5 min, the reaction was quenched with potassium carbonate(50 ml of a 5% aqueous solution). After 5 min, the solvent was removedunder vacuum, the residue was dissolved in ethylacetate (150 ml) andextensively washed with water (2×200 ml each), potassium carbonate(2×100 ml each of a 5% aqueous solution), cold sodium hydroxide (8×100ml each of a 0.5N aqueous solution), and saturated sodium chloride(2×100 ml each). The organic layer was then dried over sodium sulfateand the solvent removed under vacuum to give the product as an oil (7.9g).

TLC Analysis: A TLC plate (same type as above) was developed with 5%methanol and 1% triethylamine in methylene chloride. The R_(f) of the1-0-(4,4'-dimethoxytrityl)-6-aminohexane and the carbamate adduct of1-0-(4,4'-dimethoxytrityl)-6-aminohexane and 2,2'-sulfonyldiethanol(hereinafter referred to as the carbamate adduct) was 0.2 and 0.4,respectively.

The crude carbamate adduct was purified by silica gel chromatography(column dimensions: 5.5 cm internal diameter and 8 cm length). Thesilica gel G60 was pretreated with an ethylacetate-triethylamine-hexanesolvent system (50% ethyl acetate and 0.5% triethylamine in hexane)prior to the separation. The crude carbamate adduct was dissolved inethylacetate-hexane solvent (50% ethyl acetate in hexane), loaded ontothe pretreated column, and eluted with ethylacetate-hexane (50% ethylacetate in hexane), ethyl acetate, then by methanol-ethyl acetate (10%methanol in ethyl acetate). Ten fractions were collected, and eachfraction was analyzed by TLC (see immediately below for TLC conditions)and the appropriate fractions were combined to give the product as anoil (4.3 g).

TLC Analysis: A TLC plate (same type as above) was developed with 0:5%triethylamine in ethyl acetate. The R_(f) of the carbamate adduct wasapproximately 0.3.

Example 5 Synthesis of the succinyl ester of the carbamate adduct of1-0-(4,4'-dimethoxytrityl)-6-aminohexane and 2,2'-sulfonyldiethanol

The carbamate adduct (2.0 g) from Example 4 and 4-dimethylaminopyridine(0.49 g) (Aldrich) were dissolved in methylene chloride (20 ml) underargon, and succinic anhydride (0.41 g) was added to the stirringsolution at room temperature. After 5 min, additional methylene chloridewas added (100 ml) and the solution was washed with cold citric acid(5×100 ml each of a 10% aqueous solution of citric acid) and saturatedsodium chloride solution (2×100 ml each). The washed solution was driedover sodium sulfate and the solvent was removed under vacuum to give theproduct as an oil (2.1 g).

TLC Analysis: A TLC plate (same type as above) was developed with 5%methanol and 0.5% triethylamine in methylene chloride. The R_(f) of thecarbamate adduct was 0.4 and the R_(f) of the succinyl ester of thecarbamate adduct (hereinafter referred to as the succinyl ester) was0.3.

The crude succinyl ester was purified by silica gel chromatography(column dimensions: 5.5 cm internal diameter and 8 cm length). Thesilica gel G60 was pretreated with a methanol-triethylamine-methylenechloride solvent system (1% methanol and 0.5% triethylamine in methylenechloride) prior to the separation. The crude succinyl ester wasdissolved in a methylene chloride-methanol solvent (1% methanol inmethylene chloride), loaded onto the column, and eluted with athree-step solvent gradient (200 ml of 1% methanol and 0.5%triethylamine, 200 ml of 5% methanol and 0.5% triethylamine, and 150 mlof 15% methanol and 0.5% triethylamine, each in methylene chloride).Eighteen fractions were collected, and each fraction was analyzed by TLC(see immediately below for TLC conditions). The appropriate fractions(fractions 8-13) were combined and the solvent removed under vacuum togive the product as an oil (1.32 g).

TLC Analysis: A TLC plate (same type as above) was developed with 5%methanol and 0.5% triethylamine in methylene chloride. As before, theR_(f) of the succinyl ester was 0.3.

Example 6 Attaching the succinyl ester to 3-aminopropyl controlled poreglass forming the 3'-aminolinker support

The succinyl ester (0.4 g) from Example 5 was dissolved indimethylformamide (10 ml) under argon, and an equimolar solution of1-hydroxybenzotriazole (HOBT) and2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium (HBTU) (1.14 ml ofa 0.45M solution in dimethylformamide) (Applied Biosystems Division ofthe Perkin Elmer Corporation, Foster City, Calif. (ABI)) was added tothe stirring solution, followed by the addition of diisopropylethylamine(0.14 g). After 15 min at room temperature, 3-aminopropyl-CPG (5.58 g ofmaterial having a loading of 40 μmole per gram)(ABI) was added to thestirring solution, the stirring was stopped, and the reaction wasallowed to proceed for 2.5 hrs with occasional gentle swirling. Theslurry was transferred to a medium-grit fritted funnel where the CPGsupport was washed with methylene chloride (5×20 ml), treated with acapping reagent (10 ml 0.5M N-methylimidazole in tetrahydrofuran and 10ml 10% acetic anhydride 10% 2,6-lutidine in tetrahydrofuran), andallowed to stand for 30 min. The solution was then removed and the CPGwas washed with methylene chloride (5×20 ml), then dried under vacuum togive a white solid (5.64 g).

Example 7 Synthesis of a 3'-aminohexyl polynucleotide using the solidsupport of the present invention

Synthesis of a 3'-amino polynucleotide was performed on an AppliedBiosystems 394 polynucleotide synthesizer using standard protocols andreagents (ABI). A brief description of the chemistry used by the 394polynucleotide synthesizer is provided above in the section IV titledUtility. The solid support used in the synthesis was that whosesynthesis is described in Examples 1-6 (32 mg). The base sequence of thepolynucleotide was 5'-AGC TAG CT-3'. The product was cleaved off of thesynthesis support with the terminal trityl group still attached, and wasdetermined to be approximately 80% pure by HPLC analysis.

Example 8 Attaching a fluorescent dye to a 3'-aminohexyl polynucleotide

The crude polynucleotide (20%) from Example7 in 0.1Mtriethylammoniumacetate, pH 7 (100 μl), was added to a solution of6-carboxy-fluorescein-N-hydroxysuccinimide (6-FAM) ester (1 mg in 100 μldimethyl formamide) (Research Organics, Inc., Cleveland, Ohio), followedby the addition of a 1M NaHCO₃ /Na₂ CO₃ pH 9.0 solution (100 μl). Thesolution was vortexed and allowed to stand at room temperature for 30min. The mixture was then applied to a PD-10 gel filtration column(Pharmacia, Piscataway, N.J.) and a single fraction was collected (1 mlfraction collected after the elution of a 2.5 ml void volume) to give5'-AGCTAGCT-3'-aminohexyl-6-FAM.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. Those skilled in the art of chemistry will understand thatthere are many variations of the above support reagents, and methods forsynthesis of the above support reagents, that fall within the preview ofthe present invention.

What is claimed is:
 1. A solid support reagent for the synthesis ofpolynucleotides containing a nitrogen group at the 3'-end, the solidsupport reagent having the structure: ##STR15## wherein: T is anacid-cleavable hydroxyl protecting group;Q is a linker for connectingnitrogen and oxygen; R₁ is a nitrogen substituent; R₂ through R₄ areseparately hydrogen or lower alkyl; Y is an atom which iselectronegative with respect to carbon; X₁ is a moiety which iselectronegative with respect to carbon; Z is a bond or spacer arm; and Wis a derivatized solid synthesis support capable of linking to Z.
 2. Acompound according to claim 1 wherein T is 4,4'-dimethoxytrityl,monomethoxytrityl, α-naphthyldiphenylmethyl, ortri(p-methoxyphenyl)methyl.
 3. A compound according to claim 1 wherein Tis 4,4'-dimethoxytrityl.
 4. A compound according to claim 1 wherein Q islower alkyl; or when in combination with an adjacent oxygen loweralkylene oxide; or when in combination with a nitrogen of the solidsupport reagent, Q is amide, carbamate, sulfonamide, or urea.
 5. Acompound according to claim 1 wherein Q is lower alkyl or lower alkyleneoxide.
 6. A compound according to claim 1 wherein R₂ through R₄ ishydrogen.
 7. A compound according to claim 1 wherein Y is oxygen orsulfur.
 8. A compound according to claim 1 wherein Y is oxygen.
 9. Acompound according to claim 1 wherein X₁ is sulphonyl, carbonyl,sulfoxide, perfluoro lower alkyl, or sulfonyl-, carbonyl-, sulfoxide,nitro-, cyano-, or perfluoro lower alkyl-substituted aryl.
 10. Acompound according to claim 1 wherein X₁ is sulphonyl, carbonyl,sulfoxide.
 11. A compound according to claim 1 wherein Z is, incombination with a terminal nitrogen of the solid synthesis supportderivatized with an amino-terminated linker, carbamate, urea, amide,sulfonamide, or a group of the formula: ##STR16## wherein v is aninteger between 0 and
 20. 12. A compound according to claim 1 wherein Wis CPG derivatized with an amino-terminated linker.
 13. A compoundaccording to claim 1 wherein W is porous polystyrene derivatized with anamino-terminated linker.
 14. A solid support reagent for the synthesisof polynucleotides containing a nitrogen group at the 3'-end, the solidsupport reagent comprising a compound of the formula: ##STR17## wherein:T is an acid-cleavable hydroxyl protecting group;Q is a linkerconnecting the nitrogen and oxygen; R₁ is a nitrogen substituent; R₂ andR₃ are separately hydrogen or lower alkyl; Y is an atom which iselectronegative with respect to carbon; X₂ is a moiety which iselectronegative with respect to carbon; Z is a bond or spacer arm; and Wis a derivatized solid synthesis support capable of linking to Z.
 15. Acompound according to claim 14 wherein T is 4,4'-dimethoxytrityl,monomethoxytrityl, α-naphthyldiphenylmethyl, ortri(p-methoxyphenyl)methyl.
 16. A compound according to claim 14 whereinT is 4,4'-dimethoxytrityl.
 17. A compound according to claim 14 whereinQ is lower alkyl; or when in combination with an adjacent oxygen loweralkylene oxide; or when in combination with a nitrogen of the solidsupport reagent, Q is amide, carbamate, sulfonamide, or urea.
 18. Acompound according to claim 14 wherein Q is lower alkyl or loweralkylene oxide.
 19. A compound according to claim 14 wherein R₂ and R₃are hydrogen.
 20. A compound according to claim 14 wherein Y is oxygenor sulfur.
 21. A compound according to claim 14 wherein Y is oxygen. 22.A compound according to claim 14 wherein X₂ is sulphonyl, carbonyl,sulfoxide, cyano, perfluoro lower alkyl, or sulfonyl-, carbonyl-,sulfoxide, nitro-, cyano-, or perfluoro lower alkyl-substituted aryl.23. A compound according to claim 14 wherein X₂ is sulphonyl, carbonyl,or cyano.
 24. A compound according to claim 14 wherein Z is, incombination with a terminal nitrogen of the solid synthesis supportderivatized with a nitrogen-terminated linker, carbamate, urea, amide,sulfonamide, or a group of the formula: ##STR18## wherein v is aninteger between 0 and
 20. 25. A compound according to claim 14 wherein Wis CPG derivatized with an amino-terminated linker.
 26. A compoundaccording to claim 14 wherein W is porous polystyrene derivatized withan amino-terminated linker.